Tetraspanin proteins CD9 and CD81 regulate sperm-egg fusion, cell growth and proliferation, tumor cell motility and metastasis, and other functions. CD9 and CD81 do not serve in a typical cell surface receptor-ligand binding capacity. Rather they function in the context of multicomponent proteolipid complexes known as Tetraspanin-Enriched Microdomains (TEMs). Within TEMs, CD9 and CD81 each can directly associate with Ig superfamily transmembrane proteins known as EWI-2 and EWI-F (named for conserved Glu-Trp-Ile motifs). We suggest that EWI-2 and EWI-F are major participants in many of the functions previously ascribed to CD9 and CD81. Although sharing many of the same components, CD9/CD81-EWI-2 and CD9/CD81-EWI-F complexes differ in size and density. Furthermore, CD9/CD81-EWI-2 complexes depress cell spreading, invasion and migration;are missing from many tumor cells;impair intracranial glioblastoma growth;and are associated with improved glioblastoma patient prognosis. Conversely, CD9/CD81-EWI-F complexes are upregulated on many tumor cells, enhance cell survival, and are associated with poor glioblastoma patient prognosis. Our central guiding hypothesis is that EWI-2 and EWI-F complexes function in opposition on tumor cells. Hence, our major goals regarding EWI-2 and EWI-F complexes are to i) characterize more fully their opposing cell biological functions, ii) understand their functions in the context of TEMs, and iii) examine opposing effects on tumor cells in vivo, using a human glioblastoma/nude mouse xenograft model. More specifically, in Aim 1 we will examine EWI-2 and EWI-F effects on cell motility, morphology, invasion, adhesion, and survival;and we will examine their subcellular localization. In Aim 2, we will disrupt protein palmitoylation, which plays an essential role in holding together TEMs. This will be done by removing or inhibiting the protein acyltransferase(s) needed for tetraspanin and EWI protein palmitoylation, and by mutating specific EWI-2 and EWI-F palmitoylation sites. In Aim 3, we will use a quantitative proteomics approach to identify specific protein partners that preferentially associate with EWI-2 or EWI-F. Such proteins may hold the key towards understanding their opposing functions. In Aim 4, we will investigate the contributions of EWI-2 and EWI-F cytoplasmic domains towards their functions, with a focus on key signaling pathways (involving PKC and AKT), and on specific molecular interactions. Finally, in Aim 5 we will express wild type and mutant forms of EWI-2 and EWI-F in glioblastoma cells, in order to determine effects on intracranial tumor growth. Public Health Relevance: EWI-2 and EWI-F protein complexes have opposing functions - the former retards tumor progression, while the latter enhances it. By understanding the molecular details regarding these newly discovered molecules, we will gain new insights and new tools that can be used to combat cancer. Findings will be relevant not only to brain cancer (studied here), but also for other types of cancer, as well as for normal cell functions that involve EWI-2 and EWI-F.